Dissection of non-canonical DNA damage response mechanisms controlling the senescence program

细胞衰老是一种自然的细胞衰老机制，发生在端粒功能障碍或慢性氧化应激等条件下导致的未修复的基因组DNA损伤。衰老细胞的特征是衰老相关的稳定增殖停滞(SAPA)和分泌表型(SASP)，在正常组织修复或发育过程中有助于组织重塑。我们和其他人已经证明，衰老程序是由DNA损伤反应(DDR)控制的，DDR是由DNA双链断裂(DSB)激活的信号通路的交错网络。目前尚不清楚为什么以及如何在DSB(规范反应)几分钟内激活的DDR通过细胞周期检查点迅速建立增殖抑制，但需要数天时间才能建立稳定的SAPA和SASP。为了了解早期典型DDR和潜在持续DDR之间的转换，我们建立了通过辐射或受控端粒去封顶在正常人类细胞中诱导DNA损伤诱导衰老的纵向模型。我们发现，端粒功能障碍触发了快速的焦点DDR活动，伴随着短暂的细胞周期停滞，这一过程通过DNA损伤耐受而迅速被绕过。复制的功能失调的端粒然后导致同源重组介导的端粒姐妹染色单体融合，这是下一次有丝分裂基因组不稳定性的基础。我们还发现，非典型的DDR发生在DNA损伤诱导后的几天，其特征是DDR成分的逐渐积累，如ATM和染色质上的MRN复合体。我们的假设是，SAPA和SASP需要延迟的替代DDR活性，而不是参与DNA修复和瞬时细胞周期停滞的即时典型DDR，我们称之为非典型DDR。这一研究计划的长期目标是通过识别参与这一过程的分子调控因子并评估它们对SAPA或SASP的影响来表征和定义非规范的DDR信号。我们的具体短期目标是使用有针对性和不偏不倚的战略组合来描述这一新型的非规范DDR。我们将在蛋白质和染色质/转录水平上确定非经典DDR信号的相互作用组和分子调节因子，以了解该网络在建立和维持衰老过程中的作用。了解DDR的作用和活动仍然是当前基础基因组稳定性细胞生物学的主要挑战之一，拟议的研究计划将进一步描述一个新的非规范DDR网络，该网络交织在基因组稳定性和细胞衰老的建立中。该计划将以一种促进EDI的方式吸引HQP参与这一尖端研究课题，并将培训他们具有各种多学科技能。

Cellular senescence is a natural cell aging mechanism occurring in response to unrepaired genomic DNA damage caused by conditions like dysfunctional telomeres or chronic oxidative stress. Senescent cells are characterized by a senescence¬-associated stable proliferation arrest (SAPA) and by a secretory phenotype (SASP) that contribute to tissue remodeling during normal tissue repair or development. We and others have shown that the senescence program is controlled by the DNA damage response (DDR), which is an interlaced network of signalling pathways activated by DNA double-strand breaks (DSB). It remains unclear why and how the DDR, which is activated within minutes of a DSB (canonical response), establishes proliferation arrest rapidly via cell cycle checkpoints, but takes day to establish the stable SAPA and SASP. To understand the transition between the early canonical DDR and a potentially persistent DDR we developed longitudinal models of DNA damage-¬induced senescence via irradiation or controlled telomere uncapping in normal human cells. We discovered that telomere dysfunction triggers a rapid focal DDR activity accompanied by a transient cell cycle arrest, which is rapidly bypassed via DNA damage tolerance. Replicated dysfunctional telomeres then result in homologous recombination¬-mediated telomeric sister chromatid fusions underlying next mitosis genome instability. We also showed that a noncanonical DDR occurs several days following DNA damage induction and is characterized by a progressive accumulation of DDR components like ATM and the MRN complex on the chromatin. Our hypothesis is that the SAPA and the SASP require delayed alternative DDR activities when compared to the immediate canonical DDR involved in DNA repair and transient cell cycle arrest, a phenomenon we term non¬canonical DDR. The long-term objective of this research program is to characterize and define non¬canonical DDR signaling via the identification of the molecular regulators involved in this process and the evaluation of their impact on SAPA or SASP. Our specific short-term objectives are to characterize this novel non¬canonical DDR using a mix of targeted and unbiased strategies. We will identify the interactome and molecular regulators of non-canonical DDR signaling at the protein and chromatin/transcriptional level to understand the role of this network in establishing and maintaining senescence. Understanding the role and activities of the DDR remain one of the major current challenge in fundamental genome stability cell biology, the proposed research program will further describe a novel non¬canonical DDR network interlaced within genome stability and the establishment of cell senescence. This program will attract HQP to this cutting-edge research subject in a way that promote EDI and will train them with a variety of multidisciplinary skills.